Interpretive Summary: This work describes experiments that have been conducted in an effort to better understand the mechanisms by which aflatoxins are produced by fungi. Aflatoxins are toxic and carcinogenic compounds often produced by the fungi, Aspergillus flavus and A. parasiticus, during growth on crops such as corn, peanuts, cottonseed and treenuts. Because of the potential health risks, aflatoxin contamination of food and feed crops is also of great economic importance to farmers who cannot sell their crops due to strict domestic and international regulatory guidelines with regards to aflatoxin contamination. This study looked at the location of specific enzymes involved in aflatoxin production within the fungal cell. By determining where these enzymes are present in the cell, it may be possible to devise strategies for interrupting a critical step in aflatoxin production, thus eliminating aflatoxin contamination of food and feed crops.

Technical Abstract:
The biosynthesis of aflatoxin in Aspergillus parasiticus is a complex process that involves the activities of at least 18 pathway enzymes. The distribution of these enzymes within fungal colonies and fungal cells is not clearly understood. The objective of this study was to investigate the distribution and subcellular location of Nor-1, Ver-1 and OmtA, which represent early, middle and late enzymatic activities, respectively, in the aflatoxin biosynthetic pathway. The distribution of these three enzymes within A. parasiticus SU-1 was analyzed in time-fractionated, 72-h fungal colonies (fraction 1, 48-72 h; fraction 2, 24-48 h; fraction 3, 0-24 h). Western blot analysis and immunofluorescence microscopy demonstrated the highest abundance of Nor-1, Ver-1 and OmtA in colony fraction 2. Fungal tissues in this fraction were analyzed by immunoelectron microscopy. Nor-1 and Ver-1 were primarily localized to the cytoplasm, suggesting that they are cytosolic enzymes. OmtA was also detected in the cytoplasm. However, in cells located near the basal (substrate) surface of the colony, OmtA was predominantly detected in organeles tentatively identified as vacuoles. The role of this organelle in toxin biosynthesis is unclear. The relative distribution of OmtA to the cytoplasm or to vacuole-like organelles may depend on the age and/or physiological condition of the fungal cells.